Within the higher eukaryotic chromosome, DNA is folded into multiple levels of organization, yielding alinear compaction of DNA of approximately 20,000 fold. The highest levels of chromatin folding, consisting of the folding of 30 nm chromatin fibers, account for up to a 500:1 packing ratio and involve the organization of entire transcription and replication functional domains, their compaction within mitotic and interphase chromatids, and their architecture within interphase nuclei. Poorly characterized, it is this large-scale chromatin structure which is the focus of our research. Our long term objectives are to determine the basic folding motifs underlying large-scale chromatin structure, the functional significance of specific conformational changes associated with transcription and replication, and the underlying mechanisms which regulate these conformational transitions. Our specific aims are to address the following questions: 1) What structural motifs underlie large-scale chromatin structure of interphase chromosomes? 2) What structural rearrangments in large-scale chromatin structure occur during DNA replication and daughter chromatid segregation? 3) What structural motifs underlie mitotic chromosome condensation and decondensation? 4) Which aspects of large-scale chromatin structure require condensin or cohesin function? This proposed research will provide a basic description of the folding motifs underlying large-scale chromatin and chromosome organization. Future directions of our work will focus on integrating structural models of large-scale chromatin organization with the underlying biochemistry and molecular biology. In particular we are interested in exploring the mechanistic links between large-scale chromatin structure and nuclear architecture to transcription, replication and recombination. Knowledge gained from this research should provide valuable insight into regulation of basic molecular processes and may serve to guide design of future gene therapy vectors and stable transgene expression.